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Archives of Toxicology (v.74, #11)

No Title by Hermann M. Bolt; Petra Janning; Horst Michna; Gisela H. Degen (pp. 649-662).

A novel concept – the hygiene-based margin of safety (HBMOS) – is suggested for the assessment of the impact of potential endocrine modulators. It integrates exposure scenarios and potency data for industrial chemicals and naturally occurring dietary compounds with oestrogenic activity. An HBMOS is defined as a quotient of estimated daily intakes weighted by the relative in vivo potencies of these compounds. The Existing Chemicals Programme of the European Union provides Human and Environmental Risk Assessments of Existing Chemicals which include human exposure scenarios. Such exposure scenarios, along with potency estimates for endocrine activities, may provide a basis for a quantitative comparison of the potential endocrine-modulating effects of industrial chemicals with endocrine modulators as natural constituents of human diet. Natural phyto-oestrogens exhibit oestrogenic activity in vitro and in vivo. Important phyto-oestrogens for humans are isoflavones (daidzein, genistein) and lignans, with the highest quantities found in soybeans and flaxseed, respectively. Daily isoflavone exposures calculated for infants on soy-based formulae were in the ranges of 4.5–8 mg/kg body wt.; estimates for adults range up to 1 mg/kg body wt. The Senate Commission on the Evaluation of Food Safety (SKLM) of the Deutsche Forschungsgemeinschaft has also indicated a wide range of dietary exposures. For matters of risk assessment, the SKLM has based recommendations on dietary exposure scenarios, implying a daily intake of phyto-oestrogens in the order of 1 mg/kg body wt. On the basis of information compiled within the Existing Chemicals Programme of the EU, it appears that a daily human exposure to nonylphenol of 2 µg/kg body wt. may be a worst-case assumption, but which is based on valid scenarios. The intake of octylphenol is much lower, due to a different use pattern and applications, and may be neglected. Data from migration studies led to estimations of the daily human uptake of bisphenol A of maximally 1 µg/kg body wt. On the basis of comparative data from uterotrophic assays in rats, with three consecutive days of oral applications involved, and taking the natural phyto-oestrogen daidzein as reference (=1), relative uterotrophic activities in DA/Han rats follow the sequence: daidzein = 1; bisphenol A = 1; p-tert-octylphenol = 2; o,p'-DDT = 4; ethinyl oestradiol = 40,000. The derived values from exposure scenarios, as well as these relative potency values and bridging assumptions, led to calculations of HBMOS as a quantitative comparison of potential endocrine-modulating effects of industrial chemicals with those of natural constituents of human diet. HBMOS estimates for nonylphenol ranged between 250 and 500, dependent on bridging assumptions, and around 1000 for bisphenol A. The derivations of HBMOS were in full support of the conclusions reached by the SKLM of the Deutsche Forschungsgemeinschaft. The estimated HBMOS values for the industrial chemicals (nonylphenol, bisphenol A) appear sufficiently high to ensure the absence of a practical risk to human health under the present exposure conditions.

Keywords: Endocrine modulators Hormonally active agents Endocrine disruptors Nonylphenol p-tert-Octylphenol Bisphenol A o,p'-DDT Daidzein Genistein HBMOS Hygiene-based margin of safety

No Title by Gy.A. Csanády; J.G. Filser (pp. 663-672).

Inhalation is the most important route of absorption for many volatile substances. The inhaled chemical is distributed via the bloodstream into the organs and tissues. It is eliminated mainly unchanged by exhalation and also via metabolism. The blood concentration can be considered as a surrogate for the body burden of the chemical. It depends on the rate of uptake and on the rate of elimination. The rate of uptake by inhalation is determined by the blood:air partition coefficient of the gaseous compound, the actual concentration of the chemical already in the blood entering the lungs, the blood flow through the lungs, and the alveolar ventilation. The latter is greatly influenced by physical activity, which thus has a crucial impact on the rate of uptake. Consequently, the blood concentration of an inhaled chemical and the resulting alveolar retention, representing the rate of metabolism at steady-state, are dependent on the intensity of physical work. Both parameters can be calculated for steady-state conditions using simple algebraic equations, if one assumes that the rate of metabolic elimination is limited by the blood flow through the metabolizing organs. This assumption is valid for many rapidly metabolized inhaled gases and vapours at low concentrations present under workplace conditions. The derived equations give the theoretical background for the observations presented from a series of experimental studies which demonstrate that physical activity can be a major determinant of the toxicokinetics of inhaled compounds. Practical examples illustrate the procedure. We conclude that workplace-related physical activity should be taken into account for compounds with blood:air partition coefficients above 6 in the determination of occupational limit concentrations in air.

Keywords: Toxicokinetics Inhalation Physical activity Alveolar retention Body burden Volatile compounds

No Title by D. Ligocka; A. Sapota; K. Rydzynski (pp. 673-679).

The organ and tissue distribution, excretion and metabolism of [³H]1,2,4,5-tetramethylbenzene ([³H]durene) in male Wistar albino rats were investigated following a single i.p. administration (40 mg/kg) and within 9 days after five daily repeated administrations. Urine proved to be the main route of tritium excretion. Within the first 24 h after a single administration 69% of the radioactivity was excreted in the urine and only 9% in the feces. The highest level of tritium binding was found in the fat tissue, liver, kidneys and adrenal glands. The accumulation of tritium in the plasma proceeded with a kinetic constant of 0.49 h^–1, whereas the half-life of radioactivity decayamounted to about 6.3 h. In erythrocytes, the tritium level was found to be about three times lower than in blood plasma. The total amount eliminated during the 9 days following repeated administration was about 94% of the five doses given. The highest level of tritium was found in fat tissue and adrenal glands, followed by the liver, kidneys, sciatic nerve and muscle. A gradual decline in tritium levels was observed during the following 4 days in most tissues to reach about 2% of the dose given. The main urinary metabolites resulting from the administration of durene were 2,4,5-trimethylbenzyl alcohol (about 22%), 4,5-dimethyl-1,2-benzdialdehyde (about 19%), 2,4,5-trimethylbenzaldehyde (about 19%) and 2,4,5-trimethylbenzoic acid (about 16%). The oxygen-containing metabolites accounted for almost 80%, whereas sulphur-containing metabolites accounted for approximately 10% of the products of biotransformation. In conclusion, most of the durene administered has a relatively rapid turnover rate, with minor levels retained in the tissues for longer time periods.

Keywords: Durene-3H Distribution Excretion Metabolism Rats

No Title by P. Begemann; R.J. Srám; H.-G. Neumann (pp. 680-687).

1,3-Butadiene (BD) is an important chemical widely used in the synthetic rubber industry. Hemoglobin adducts of two of its reactive metabolites have been already investigated as possible parameters for exposure assessment. In this study hemoglobin adducts of epoxybutene (EB) were analyzed in blood samples from 17 workers in a BD monomer production unit and 19 controls in a heat production unit of a petrochemical plant near Prague, Czech Republic. BD exposure was determined by personal air sampling. The median level of exposure was 440 µg/m³ (range <11–17 mg/m³) for the exposed workers and <6 µg/m³ (<5–150 µg/m³) for the controls. The adduct N-(2-hydroxy-3-butenyl)valine (HBVal) formed by the reaction of the N-terminal valine of globin with carbon-1 of EB was measured. The N-alkylated amino acid was analyzed by gas chromatography/mass spectrometry (GC/MS) after degradation by the modified Edman procedure. Using published methods problems arose with high background levels, especially in the negative ion chemical ionization (NCI) mode. In the present study a limit of detection of 0.2 pmol/g globin was achieved by using 400 mg globin, a variation in extraction solvents, an additional purification step and a widely extended GC temperature program. The median hemoglobin adduct level of the Czech BD monomer production workers (0.7 pmol/g globin; n=17) was significantly higher than that of the controls (0.2 pmol/g globin; n=19; P<0.05). Smoking controls showed higher hemoglobin adduct levels than nonsmoking controls (P<0.1) and significantly higher BD exposure levels (P<0.01).

Keywords: Biomonitoring 1,3-Butadiene Epoxybutene Hemoglobin adducts

No Title by Evert H. Delbanco; Hermann M. Bolt; Wolfgang W. Huber; Sonja Beken; Frank Geller; Stathis Philippou; Frank H. Brands; Thomas Brüning; Ricarda Thier (pp. 688-694).

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugation with glutathione. It has mostly been assumed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n=21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST µ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Keywords: Glutathione transferases Kidney tissue Renal carcinomas Phenotyping Genotyping

No Title by Bernard Fauconneau; Sabrina Stadelmann-Ingrand; Sylvie Favrelière; Julien Baudouin; Laurent Renaud; Alain Piriou; Claude Tallineau (pp. 695-701).

Astrocytes are known to play a key role in buffering extracellular pH variations and, in addition, they are particularly resistant to oxidative stress and subsequent lipid peroxidation. This great resistance may be ascribed to the presence of high concentrations of certain antioxidants, but another explanation may be the presence of a high quantity of plasmalogens, which are a special group of glycerophospholipids characterized by a vinyl ether bond instead of an ester bond in the sn-1 position of the glycerol backbone. Plasmalogens are sensitive to free radical attack and acidity, and numerous works have supported the hypothesis that they may be antioxidant molecules that protect cells from oxidative stress. The aim of this work was to investigate, on astrocytes in lactic acid-induced oxidative stress (pH 5.5), the behavior of phospholipids and, in particular, plasmalogens. Two main techniques, based on the susceptibility of the vinyl ether bond to hydrolysis, were employed in this study to measure plasmalogen levels. In both cases, the sn-1 vinyl ether linkage was cleaved using mercuric chloride, producing a lysophospholipid that was assessed by phosphorus measurement or using HCl treatment, producing a long-chain fatty aldehyde assayed by gas chromatography/mass spectrometry. On astrocytes in culture, only plasmenylethanolamine (PlmEtn) was evidenced, representing 40% of glycerophosphoethanolamine lipids. When astrocytes were incubated with lactic acid, no modification in the amount of PlmEtn was seen. Furthermore, free aldehydes and aldehydes corresponding to the quantity of intact plasmalogens were similar to those observed on controls. In addition, the constancy of two lipid peroxidation markers, thiobarbituric acid reactive substances and polyunsaturated fatty acids, was clear evidence of the resistance of these cells in lactic acid conditions. In conclusion, our results fail to demonstrate a major role of plasmalogens in the resistance of astrocytes in lactic acid-induced oxidative stress.

Keywords: Astrocytes Lactic acid Plasmalogens Oxidative stress Fatty acids

No Title by Andrzej Dekundy; Piotr Blaszczak; Rafal Kaminski; Waldemar A. Turski (pp. 702-708).

Both organophosphate (OP) and carbamate pesticides may produce seizures and death commonly attributed to the inhibition of acetylcholinesterase (AChE) and subsequent excess of acetylcholine (ACh). The anticonvulsant and neuroprotective properties of N-methyl-D-aspartate (NMDA) receptor antagonists in animals encouraged us to investigate their effects on the toxic and convulsant properties of OP and carbamate pesticides. Adult Swiss mice were systemically injected with the OP pesticide, chlorfenvinphos (CVP), or the carbamate pesticide, methomyl (MET). Both CVP and MET induced dose-dependent seizure activity and death in mice. Pretreatment with the muscarinic antagonist, atropine (ATR), at a dose of 1.8 mg/kg did not prevent seizures but decreased the lethal effects of CVP and MET. Pretreatment with the NMDA antagonists, dizocilpine (MK-801) at a dose of 1 mg/kg or 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at a dose of 10 mg/kg, influenced neither MET-induced seizures nor CVP- or MET-induced death. However, both MK-801 and CPP blocked CVP-induced seizures. Concurrent administration of ATR and the NMDA antagonists prevented seizures produced by CVP, but not those produced by MET. Nevertheless, both MK-801 and CPP coadministered with ATR markedly enhanced its antilethal effects in CVP- and MET-intoxicated mice. The antidotes had no influence upon brain AChE activities in mice treated with saline or CVP or MET. It seems that combined treatment with ATR and NMDA receptor antagonists might be of clinical relevance.

Keywords: Organophosphate Carbamate NMDA antagonist Atropine Mice

No Title by Alexius Freyberger; Elke Hartmann; Heinrich Hildebrand; Franc Krötlinger (pp. 709-715).

The stilbene derivative resveratrol (RES) is a phytoalexin of grapes, peanuts and other fruits. It is structurally related to stilbene estrogens and an estrogenic potential of RES has recently been demonstrated in a number of in vitro studies. In this investigation, the uterotrophic responses of immature Wistar rats to subcutaneous administration of RES (18, 58, and 575 mg/kg) and the reference estrogen ethinylestradiol (EE2; 0.3, 1, 3, 30 µg/kg) on three consecutive days were determined. Uterine weight, histopathological changes, immunohistochemical expression of nuclear estrogen receptor-α (ERα) and progesterone receptor (PR) protein, gene expression of ERα and PR at the messenger ribonucleic acid (mRNA) level and peroxidase induction were examined. EE2 dose dependently increased uterine weight, enlarged the uterine lumen and induced hypertrophy of epithelial, stromal and myometrial cells. Expression of ERα protein in epithelial, stromal and myometrial nuclei and of PR protein in epithelial nuclei was reduced in EE2-treated rats, while PR protein in stromal and myometrial nuclei was increased in a dose-dependent manner. EE2 increased messenger ribonucleic acid (mRNA) levels of uterine PR and induced peroxidase activity. In contrast, RES rather mildly decreased uterine weight, while histology did not reveal differences between controls and RES-treated rats. Expression of nuclear ERα protein was dose dependently decreased in epithelial, stromal and myometrial cells of RES-treated rats, while nuclear PR protein content was similar in controls and RES-treated rats. Following administration of RES, a trend toward reduced levels of ERα and PR mRNA was found, while no peroxidase induction occurred. Plasma levels of RES, 45 min after the administration of a single subcutaneous dose of 500 mg/kg, were in the range 1–2 µM. In summary, an estrogenic potential of RES could not be substantiated in this in vivo study, although the most effective route of administration and extremely high doses were used and plasma levels were in the range reported to be effective in vitro. Whether other pharmacological properties of RES could mediate the observed changes in RES-treated animals is discussed.

Keywords: Resveratrol Ethinylestradiol Uterotrophic assay Estrogen receptor Progesterone receptor

No Title by Frank Seiler; Bernd Rehn; Stefanie Rehn; Joachim Bruch (pp. 716-719).

Exposure to silica can lead to fibrosis and the development of lung tumors in the rat. Based on these animal studies and on epidemiological data, silica has been classified as a human carcinogen. The initial mechanisms have not been finally clarified, but particle-induced tumor formation is at least closely associated with inflammation, the production of reactive oxygen species (ROS) and DNA damage. We investigated the dose-dependent effects of silica on the formation of the major DNA oxidation product 8-oxoguanine (8-oxoGua) in rat lung cells, on p53 (p53) and p53 mutant protein (p53 mut) synthesis, as well as on the amount of the surfactant phospholipids phosphatidylinositol (PI) and phosphatidylglycerol (PG) in the bronchoalveolar lavage fluids (BALF) as indicators of fibrotic processes in the lung. Rats were exposed by intratracheal instillation to various amounts of DQ12 quartz (0.15, 0.3, 0.6, 1.2, 2.4 mg/animal) and lungs were investigated after 21 and 90 days. PG decreased and PI increased quartz dose dependently. 8-oxoGua was significantly increased only after 1.2 and 2.4 mg quartz/animal. Cells expressing p53 protein were increased at 1.2 and 2.4 mg, p53 mutant protein only at 2.4 mg/animal. This indicates a no-effect level for mutagenicity at a low, but still fibrogenic quartz exposure.

Keywords: Quartz Rat 8-Oxoguanine Mutagenicity Fibrogenicity No-effect level

No Title by Lakshmi K. Akkineni; Magnus Zeisig; Pawel Baranczewski; Lars-Gösta Ekström; Lennart Möller (pp. 720-731).

The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by ³²P-postlabeling and ³²P-high-performance liquid chromatography (³²P-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250°C and a peak around a distillation point of approximately 400°C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16 substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The ³²P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.

Keywords: Bitumen Petroleum Coal tar DNA adducts 32P-HPLC Human

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Archives of Toxicology (v.74, #11)